Systems, Devices, and Methods for Automatic Cell Sorting

This application claims priority to U.S. Provisional Patent Application Ser. No. 63/520,861 filed Aug. 21, 2023, the content of which is incorporated herein by reference in its entirety for all purposes.

The present disclosure relates to systems, devices, and methods for cell sorting, for example, automated magnetic cell sorting.

Cell therapies involve collecting cells from an individual, processing the cells, and utilizing the processed cells to achieve a clinical response in the same or a different individual. Cell processing (e.g., growing or culturing cells) is a complex workflow that involves multiple steps, including sorting the cells to separate targeted cells from non-targeted material. Sorting cellular material using a cell sorting system can be performed by tagging targeted cells with magnetic particles, such that the targeted cells may be separated from the non-targeted cells by using a magnet to attract the targeted cells. The magnet is typically brought into close proximity with the cellular material manually, which is operationally inefficient and labor intensive. Typical cell sorting systems can only accommodate magnetic particles that are either micrometer-sized or nanometer-sized, so using inappropriately sized magnetic particles may clog the flow paths through the system. Even cell sorting systems with appropriately sized magnetic particles and/or components frequently experience clogging, which generally result in cell systems being single use as the components often cannot be rinsed or washed to release the clogged particles. In addition to sizing the components based on the magnetic particle size, cell sorting systems typically require advanced knowledge of the total throughput of cellular material in order to acquire components with volumes configured to accommodate the total throughput. Accordingly, additional systems and methods for cell sorting are desirable.

The present disclosure relates generally to systems, devices, and methods for cell sorting within an automated cell processing system. In general, an automated cell sorting system may include a cartridge having a cell sorting module and an instrument within a bay of a cell processing workcell. The cell sorting module may include a flow cell. The flow cell may be configured to hold a volume of between about 1 mL to about 15 mL. The instrument may include a magnetic array that may be couplable to the flow cell. Each of the magnets within the magnetic array may have a width of w and may be spaced apart by between about w/3 to about ¾w, about w/3 to about ⅔w, about ⅖w to about ⅗w, or about w/2. The magnets of the magnetic array may be arranged with alternating polarities proximate to the flow cell. The magnetic array may be coupled to an actuator of the instrument. The actuator may be configured to translate the magnetic array. In some variations, the actuator may comprise a piston. The instrument may further include a sensor configured to measure a parameter of the cell sorting module. The cell sorting module may further include a purge line.

In some variations, the flow cell may include a film. The film may have a thickness of about 100 microns to about 500 microns. The flow cell may have a height of between about 1/12w to about ⅛w. The height of the flow cell may be between about 0.25 mm and about 3 mm. In some variations, the height of the flow cell may be about 1.5 mm.

In some variations, the cartridge may further include one or more additional modules selected from the group consisting of a bioreactor module, an electroporation module, an elutriation module, and a spinoculation module. The cell sorting module and the one or more additional modules may be fluidically connected. The cell processing workcell may include a robot configured to move the cartridge to a second bay.

Also described herein are methods directed to automatically sorting cells. A method for automated cell sorting may include coupling a cell sorting module within a cartridge to a magnetic array of an instrument within a workcell and flowing a cell suspension through a flow cell of the cell sorting module in batches. The cell suspension may include cells tagged by micrometer-sized or nanometer-sized magnetic particles, providing flexibility to the overall system. Each batch may be maintained within the flow cell for between about 3 and about 6 minutes. In some variations, the batch of the cell suspension may have a volume of between about 8 mL to about 10 mL. When nanometer-sized magnetic particles are used, they may have a diameter between about 50 nanometers to about 150 nanometers. When micrometer-sized magnetic particles are used, they may have a diameter between about 1 micron and about 6 microns. In some variations, between 1 to 20 batches may be flowed through the flow cell.

The method may further include decoupling the magnetic array by retracting an actuator coupled to the magnetic array, monitoring cell stiction within the flow cell using a prism and a sensor within the instrument, and/or purging the flow cell using a purge line. In some variations, decoupling the magnetic array, monitoring cell stiction, and/or purging the flow cell may be performed automatically. The method may still further include automatically transferring the magnetically tagged cells to a second module within the cartridge in accordance with a predefined workflow: The second module may be configured to perform a second cell processing step.

In some variations, a method for automated cell sorting may include moving a cartridge including a cell sorting module from a feedthrough to a bay of a workcell. The workcell may include an instrument having a magnetic array coupled to an actuator. The method may further include translating the magnetic array via the actuator to engage the cell sorting module and flowing a cell suspension through a flow cell of the cell sorting module in batches. The cell suspension may include cells that are tagged with either micrometer-sized or nanometer-sized magnetic particles. In some variations, the cell suspension may include cells that are tagged with both micrometer-sized and nanometer-sized magnetic particles.

Additional embodiments, features, and advantages of the invention will be apparent from the following detailed description and through practice of the invention.

Disclosed herein are devices, systems, and methods for sorting cells in a cell processing system. The sorting of cells may include sorting a cell suspension, which may separate cells from other material in the cell suspension. Cell sorting may be performed by a cell sorting system, which may include one or more cell sorting modules within a cartridge and one or more cell sorting instruments within a bay of a cell processing workcell. The cell sorting module is couplable to the cell sorting instrument for performing the step of cell sorting. The cell sorting system may utilize magnetic particles coupled to cells of a pre-determined type in the cell suspension. The magnetic particles may facilitate positive or negative sorting. In positive sorting, the magnetic particles may couple to cells intended for further processing (e.g., targeted cells). The targeted cells may be separated from the non-targeted cells via one or more magnets that attract the magnetic particles, such that the non-targeted cells may be removed from the cell sorting system and discarded. In negative sorting, the magnetic particles may couple to cells that are not intended for further processing (e.g., non-targeted cells). The non-targeted cells may be separated from the targeted cells via one or more magnets that attract the magnetic particles, such that only the targeted cells may continue through the cell sorting system for further processing. The cell sorting system described herein may be configured to perform either positive or negative sorting, and is configured to sort nanometer-sized and/or micrometer-sized magnetic particles.

Accordingly, a batch of the cell suspension may flow through a cell sorting module within the cartridge comprising a flow cell. A magnetic array of a cell sorting instrument within a cell processing workcell may be engaged with (e.g., in contact with, proximate to) the cell sorting module, where the cell sorting instrument comprises a magnetic array. The magnetic array may be configured to attract the magnetic particles coupled to targeted cells. The targeted cells coupled to the magnetic particles (e.g., magnetically tagged cells) may be pulled towards the magnetic array such that the targeted cells may remain stationary within the flow cell while the non-targeted material (e.g., media, non-targeted cells, buffer) may continue to flow through the flow cell. The magnetic array may be disengaged from the cell processing module of the cartridge once the batch of cell suspension has been completely flown through the flow cell. The non-targeted material may be removed from the flow cell such that only the targeted cells remain within the flow cell. The targeted cells, which may be successfully sorted, may then be removed from the flow cell. The cell sorting module of the cartridge may be configured to transfer the sorted cells to another module of the cartridge (e.g., by being fluidically coupled to a second module), such that one or more additional cell processing steps may be performed on the cells within the cartridge.

The cell sorting module described herein may be configured to repeatedly perform cell sorting automatically with micrometer- and/or nanometer-sized magnetic particles. For example, the magnetic particles may be added to the cell suspension and subsequently flowed through the cell sorting module automatically in accordance with a predetermined workflow. The ability to sort both micrometer-sized and nanometer-sized magnetic particles is a unique feature that is not available in predicate systems, which are typically configured to work with only micrometer-sized particles or nanometer-sized particles, but not both. In another example, the magnetic array may engage and disengage the flow cell automatically in accordance with a predetermined workflow, which may facilitate more efficient cell sorting and eliminate manual intervention by a user. Furthermore, the cell sorting modules described herein may be configured to be rinsed or washed, such that the cell sorting module may be reused. For example, the cell sorting module may be fluidically connected to a fluidic manifold configured to introduce one or more cleaning agents into the cell sorting module after sorting a batch of cell suspension. Accordingly, multiple batches of cell suspension may be sorted in series. The flow cell of the cell sorting module may be configured to accommodate magnetic particles of varying sizes (e.g., micrometer- and/or nanometer-sized) without becoming clogged. The size of the magnetic particles may vary between batches. Additionally, the cell sorting module may be configured to remove any cells that may become stuck within the cell sorting module. For example, the cell sorting module may be configured to introduce a fluid (e.g., air bubbles) after disengaging the magnetic array from the flow cell. The fluid may be configured to remove any targeted cells that may be adhered (e.g., stuck) to the flow cell (e.g., cell stiction). That is, the fluid may apply a force of a magnitude greater than a stiction force (e.g., a capillary force, an electrostatic force, a van der Waals force, a residual stress) that is attracting the stuck cells to the flow cell. The force applied by the fluid to the stuck cells may be determined by one or more of a flow rate, velocity, volume, density, pressure, temperature, and humidity of the fluid. The force may continue to be applied until all of the stuck cells are released. Cell stiction may be monitored automatically by a sensor (e.g., camera) coupled to the cell sorting instrument within the workcell. Accordingly, the cell sorting modules and corresponding cell sorting instruments described herein may be configured to automatically and repeatedly perform high-throughput cell sorting using magnetic particles of various sizes.

The cell processing systems described herein may be configured to perform one or more cell processing steps in a workcell. The workcell may comprise a closed, automated environment, which may be configured to maintain a sterile environment. The workcell may receive a cartridge and perform one or more cell processing steps on cells in a cell solution (e.g., cell suspension) contained within the cartridge. For example, the cell processing system may comprise a workcell comprising a plurality of instruments that may each be configured to independently perform one or more cell processing steps to the cells and/or cell solution, and a robot capable of moving the cartridge within the workcell (e.g., between one or more bays). The robot and/or instruments may be configured to automatically operate such that operator assistance may not be required at any point during the workflow. For example, the robot may receive the cartridge and move the cartridge between locations (e.g., instruments, bays, storage vaults, feedthroughs) within the workcell according to a pre-programmed workflow, where each location may be associated with one or more cell processing steps. After performing one or more cell processing steps of the pre-programmed workflow, the workcell may be configured to transfer the cartridge out of the workcell (e.g., via the robot). Additionally, or alternatively, at least a portion of the cell solution may be transferred (e.g., via a fluid device or a fluidic manifold) to a second cartridge.

The cell solution (e.g., cell suspension) described herein may contain cells that may be processed for subsequent use in cell therapies. The cell solution may comprise cells (e.g., allogeneic cells) in a fluid, such as a media (e.g., cell culture media). The cell solution may contain cells from the same or different donors. Cells from the same donor may be split between one or more cartridges, such that separate cell processing steps may be performed on each cartridge and increase the overall throughput of the cell processing system described herein. The cell solution may be transferred to the cartridge prior to loading the cartridge into the workcell, such as by operating personnel. In some variations, the cartridge may be empty when loaded into the workcell such that the workcell may transfer a cell solution to the cartridge. In some variations, the cells from two or more cartridges may be combined according to a pre-determined ratio, which may correspond to an intended therapeutic treatment for a patient.

An illustrative cell processing system for use with the automated devices, systems, and methods is shown in. Shown there is a block diagram of a cell processing systemcomprising a workcelland controller. The workcellmay comprise one or more of an instrument, a robot(e.g., robotic arm), a reagent vault, a sterile liquid transfer port, a sterilant source, a fluid source, a pump, and a sensor(s). A cartridgeand a fluid device, which may be provided outside of the workcelland used within the workcell, are illustrated in dashed lines. In some variations, the fluid devicemay be a sterile liquid transfer device (SLTD). However, it should be appreciated that the fluid devicemay be configured to transfer any fluid (which includes liquids), whether sterile or not. The controllermay comprise one or more of a processor, a memory, a communication device, an input device, and a display.

The workcellmay comprise a fully, or at least partially, enclosed housing inside which one or more cell processing steps may be performed in a fully, or at least partially, automated process. The cartridgemay be moved using the robotto reduce manual labor in the cell processing steps, and fluid transfers into and out of the cartridgemay also be performed in a fully or partially automated process, as will be described in detail herein. For example, one or more fluids may be stored in a fluid device, such that the one or more fluids may be transferred to the cartridgeand/or removed from the cartridgevia the fluid device. In some variations, the fluid devicemay be moved within the systemby the robot. Accordingly, the workcelldescribed herein advantageously enables the transfer of fluids in an automated and metered manner for automating cell therapy manufacturing.

The workcellmay facilitate fluid transfers and/or cartridge transfers. For example, in some variations, the robotmay be configured to move more than one cartridgebetween different bays to perform a predetermined sequence of cell processing steps (e.g., workflow). In this way, multiple cartridgesmay be processed in parallel, as different steps of the cell processing workflow may be performed at the same time on different cartridges. In another example, a sterile liquid transfer portmay be coupled between two or more cartridgesto transfer a cell product and/or other fluid between the cartridges. Furthermore, the sterile liquid transfer portmay be coupled between any set of fluid-carrying components of the system(e.g., cartridge, reagent vault, fluid source, fluid device, etc.). For example, a first sterile liquid transfer port may be coupled between a first cartridge and a corresponding sterile liquid transfer port of a fluid device.

Other suitable cell processing systems and aspects thereof are provided in, e.g., U.S. patent application Ser. No. 17/198,134, published as U.S. Patent Publication No. 2021/0283565, U.S. patent application Ser. No. 18/731,095, U.S. patent application Ser. No. 18/759,602, and U.S. patent application Ser. No. 18/807,699, the content of each of which is incorporated in its entirety by reference herein.

The cell processing systems described herein may comprise one or more cartridges having one or more modules configured to interface with, or releasably couple to, one or more instruments within the workcell. Some or all of the modules may be integrated in a fixed configuration within the cartridge, though they need not be. Indeed, one or more of the modules may be configurable or moveable within the cartridge, permitting various formats of cartridges to be assembled. For example, the cartridge may be a single, closed unit with fixed components for each module, or the cartridge may contain configurable modules coupled by configurable fluidic, mechanical, optical, and electrical connections. In some variations, one or more sub-cartridges, each containing a set of modules, may be used to perform various cell processing workflows. The modules may each be provided in a distinct housing or may be integrated into a cartridge or sub-cartridge with other modules. The disclosure generally shows modules as distinct groups of components for the sake of simplicity, but it should be noted that these modules may be arranged in any suitable configuration. For example, the components for different modules may be interspersed with each other such that each module may be defined by the set of connected components that collectively perform a predetermined function. However, the components of each module may or may not be physically grouped within the cartridge. In some embodiments, multiple cartridges may be used to process a single cell product through transfer of the cell product from one cartridge to another cartridge of the same or different type and/or by splitting cell product into more cartridges and/or pooling multiple cell products into fewer cartridges.

Generally, each of the instruments within the workcell interfaces with, or is releasably coupled to, its respective module or modules on the cartridge in order to carry out a specific cell processing step. For example, when a cartridge has a cell sorting module, it may be moved by the robot to a bay within the workcell having a cell sorting instrument so that the cell sorting module may be coupled to the cell sorting instrument in order to sort the cells within the cartridge. One advantage of such split module/instrument designs is that expensive components (e.g., motors, sensors, heaters, lasers, etc.) may be retained in the instruments of the system while less expensive components may reside in the cartridge.

As illustrated in, the cartridgemay be configured to contain (e.g., house) a cell solution (e.g., cell suspension) for cell processing. Any number of cell processing steps may take place upon the cells within the cartridge. Accordingly, the cartridgemay comprise one or more of a bioreactor, an electroporation module, an elutriation module, a spinoculation module, a cell sorting module, and a fluidic manifold. In instances where cell sorting is to be performed, specific reagents (e.g., magnetic particles) may be added to a cell solution within one or more of the cartridge molecules. The magnetic particles are configured to couple to cells of a specific type (e.g., targeted cells) as described above. The elutriation modulemay be configured to perform an elutriation process, wherein cellular material may be separated according to size, shape, and/or density. The spinoculation modulemay be configured to perform a spinoculation process, wherein cells of different types may be bound together.

The fluidic manifoldmay be configured to transfer one or more fluids between one or more modules of the cartridge. For example, the fluidic manifoldmay transfer a cell solution from the bioreactor moduleto the cell sorting module. The cell solution may include cellular material, including targeted cells coupled to magnetic particles. In another example, the fluidic manifoldmay transfer a cell solution from the cell sorting moduleto any other module, such as after a cell sorting process has been performed. The fluidic manifoldmay be configured to transfer the sorted cells (e.g., targeted cells) to one module and non-targeted material to a different module.

Other suitable cartridges and cell processing modules that may be used with the automatic cell processing work cells described herein are provided in, e.g., U.S. patent application Ser. No. 18/652,602. U.S. patent application Ser. No. 18/532,621. U.S. patent application Ser. No. 18/620,826, and U.S. patent application Ser. No. 18/611,632, the content of each of which is incorporated in its entirety by reference herein. Other suitable sampling systems and devices are provided in, e.g., U.S. patent application Ser. No. 18/638,658, the content of which is incorporated in its entirety by reference herein.

Referring to, an illustrative variation of a cartridgeis shown. The cartridgemay comprise an elutriation module, a fluidic manifold, a first cell sorting module, a second cell sorting module, an auxiliary module, a fluid device tray, a liquid container, and a pump module. While shown in these figures as having two cell sorting modules, it should be understood that any number of cell sorting modules may be used as desirable. For example, the cartridge may contain 1, 2, 3, 4, or even more cell sorting modules depending on the size of the cartridge, the existence of other cell processing modules within the cartridge, and so on. The cell sorting modules,may perform a cell sorting process, as will be described in further detail below. The electroporation modulemay be configured to facilitate intracellular delivery of macromolecules (i.e., transfection by electroporation). An electrical discharge from one or more capacitors, or current sources, may generate sufficient current in the chamber to promote transfer of a polynucleotide, protein, nucleoprotein complex, or other macromolecule into the cells in the cell product. The fluidic manifoldmay comprise at least one fluid conduit. The at least one fluid conduit of the fluidic manifoldmay be configured to allow fluid to pass therethrough. For example, the at least one fluid may be a liquid or a gas. In some variations, the at least one fluid may comprise a solution of cells of varying sizes and densities. The fluidic manifoldmay comprise at least one fluid inlet and at least one fluid outlet, and may comprise at least one valve. The fluidic manifoldmay be fluidically connected to at least one module within the cartridge. For example, the fluidic manifoldmay be configured to transfer at least one fluid to the first and/or second cell sorting modules,. The fluidic manifoldmay be in communication with a controller, such as the controllerdescribed in reference to. For example, at least one valve of the fluidic manifoldmay open and/or close in response to a command sent by the controllerto transfer fluid between various modules of the cartridge in accordance with a predetermined workflow. In addition to the fluidic manifold, the cartridge may also include one or more fluid conduits (e.g., tubes) to distribute fluid between the different modules.

The fluid transfer port traymay comprise one or more ports configured to transfer fluid to or from one or more fluid devices. That is, each port of the fluid transfer port traymay be configured to facilitate a sterile liquid transfer. In some variations, each port may be fluidically connected to a fluidic conduit configured to fluidically connect with at least one module of the cartridge. For example, each port of the fluid transfer port traymay be fluidically connected to the fluidic manifold. In this way, a fluid may flow from a fluid device coupled to a port of the fluid transfer port trayto the fluidic manifold, or vice versa. In some variations, each port of the fluid device traymay be fluidically connected to the liquid storage container. The liquid storage containermay be configured to contain a fluid. In some variations, the fluid may be a liquid or a gas. In some variations, the liquid storage containercomprises a plurality of liquid containers. For example, the liquid storage containermay comprise one container, two containers, or three containers. The liquid storage containermay be fluidically connected to at least one module of the cartridge. In some variations, the liquid containermay be fluidically connected to the fluidic manifold. Accordingly, a fluid may flow between a port of the fluid transfer port tray, the fluidic manifold, and the liquid storage container.

The cartridge may further comprise a pump modulehaving a pump configured to pump fluid in one or more directions along at least one fluid path. For example, the pump modulemay be configured to pump fluid to or from one or more of the elutriation module, the fluidic manifold, the cell sorting modules,, the auxiliary module, the fluid device tray, the liquid container, and any other module within the cartridge. The auxiliary modulemay be configured to engage with at least one instrument and/or module. The auxiliary modulemay comprise at least one electrical connector and/or at least one fluidic connector. In some variations, the auxiliary modulemay be removed and replaced by any other module.

Various materials may be used to construct the cartridge (including the modules thereof) and the cartridge housing, including metal, plastic, rubber, and/or glass, or combinations thereof. The cartridge, its components, and its housing may be molded, machined, extruded, 3D printed, or any combination thereof. The cartridge may contain components that are commercially available (e.g., tubing, valves, fittings). The commercially available components may be attached or integrated with custom components or devices. The housing of the cartridge may constitute an additional layer of enclosure that further protects the sterility of the cell product.

i. Cell Sorting Module

The cell sorting module within the cartridge, when coupled to a cell sorting instrument within the workcell, may be configured to perform cell sorting. The cell sorting module may comprise a flow cell, and when the cell sorting module is engaged with a magnetic array of a cell sorting instrument, cells may be flowed through the flow cell of the cell sorting module to sort the cells into targeted cells and non-targeted cells. In some variations, the cell sorting process may target cells for use in additional cell processing steps and/or cell therapies. For example, the targeted cells may comprise T-cells, which may include CD4+ and/or CD8+ cells. Accordingly, the reagents may comprise magnetic particles (e.g., magnetically conjugated beads) that correspond to the targeted cells. That is, for example, the reagents may comprise magnetic particles configured to couple to CD4+ cells, CD8+ cells, or both. Once coupled (e.g., bound, tagged) to one or more magnetic particles, the targeted cells may be referred to as magnetically tagged cells. The flow cell may be disposed in proximity to a magnetic array of the cell sorting instrument, where the magnetic array may generate a magnetic field across the flow cell to attract the targeted cells for separation, capture, recovery, and/or purification. The magnetic field may comprise magnetic field lines that extend from a north pole of the magnetic array to a south pole of the magnetic array. The magnetic field lines may extend across the flow cell such that the magnetic field interacts with the fluid flowing therethrough. For example, the magnetic array may be configured to generate a magnetic field such that a magnetophoretic force equals a drag force exerted by the fluid flowing through the flow cell. That is, the magnetophoretic force may be applied to the one or more magnetic particles coupled to targeted cells such that the targeted cells remain stationary within the flow cell while the rest of the fluid continues to flow through the flow cell. The targeted cells may form a monolayer on an inner surface of the flow cell proximate to the magnetic array, such that the fluid may not become clogged within the flow cell. The continuing flow of fluid may apply the drag force to the stationary targeted cells.

Referring to, a block diagram of an exemplary variation of a cell sorting moduleis shown. The cell sorting modulemay comprise a flow celland a purge line. The flow cellmay comprise a fluid channel, a film, an inlet port, an outlet port, and a gas port. The fluid channelmay be configured to receive a cell suspension. For example, the fluid channelmay comprise one or more sidewalls configured to retain a fluid (e.g., impermeable to liquid and gas). In some variations, the fluid channelmay comprise 1, 2, 3, or 4 sidewalls. The sidewalls may be arranged to define a channel, groove, depression, or lumen. In an exemplary variation, the fluid channelmay comprise 3 sidewalls arranged to define a channel with a rectangular cross-section. In further variations, the fluid channelmay comprise any suitable cross-sectional shape, such as a circle, a triangle, a square, or a combination thereof. The fluid channelmay comprise a length and a width, where the length may be greater than the width. The length and width of the fluid channelmay correspond to similar dimensions of a magnetic array, such as the magnetic array described further below. The fluid channel may be covered by the film, such that a cell suspension may be contained within the fluid channelwithout leaking. Accordingly, the filmmay be coupled to the body of the flow cellby an adhesive configured to form a fluid-tight seal around the fluid channel. In further variations, the fluid channeland filmmay not be separate components, such that the fluid channelmay be integrally formed with the film.

The inlet portand outlet portmay extend through the film, which may facilitate fluid flow through the fluid channel. For example, fluid (e.g., cell suspension) may flow into the fluid channelvia the inlet portand out of the fluid channelvia the outlet port. Each of the inlet portand outlet portmay be fluidically connected to the fluidic manifolddescribed in reference to. Accordingly, the fluidic manifoldmay help direct the flow in and/or out of the flow cell. The fluidic manifoldmay comprise one or more valves and/or pumps to help direct the fluid through the flow cell, which in turn may be controlled by the controllerdescribed in reference to.

The inlet portand outlet portmay be positioned at opposite ends of the fluid channelto facilitate flow of the cell suspension through the fluid channel. For example, the inlet portmay be positioned at a first end of the fluid channeland the outlet portmay be positioned at a second end of the fluid channel, relative to the longitudinal dimension (e.g., length) of the fluid channel. The relative positions of the inlet portand outlet portmay help facilitate unidirectional flow along the length of the fluid channel. Unidirectional flow of the cell suspension along the fluid channelmay help prevent clogging. In some variations, the cell sorting modulemay be oriented in an upright configuration, such that the inlet portmay be positioned higher than the outlet port, relative to a vertical dimension defined by the cell sorting module. The upright configuration of the cell sorting modulemay leverage gravity to facilitate fluid flow through the fluid channel, alone or in combination with the fluid control provided via the fluidic manifoldand pump.

The gas portmay similarly extend through the film, such that gas may flow into the fluid channel. The gas portmay be positioned at the first end of the fluid channeladjacent to the inlet port. The gas portmay be fluidically connected to the purge lineof the cell sorting module. For example, the purge linemay be configured to transfer a fluid (e.g., air bubbles, a liquid) into the fluid channel. The purge linemay be fluidically connected to the fluidic manifolddescribed in reference to. The fluid introduced by the purge linemay be configured to flow through the flow cellsuch that the fluid may interact with cells contained therein. For example, in some variations, the purge linemay perform a purging process, such as a bubble sweep. The purging process may introduce air bubbles into the flow cellto release any targeted cells that may be adhered (e.g., stuck) to one or more sidewalls of the fluid channeland/or film. That is, targeted cells may be adhered to one or more sidewalls by one or more stiction forces such as a capillary force, electrostatic force, van der Waals force, and a residual stress. The fluid (e.g., air bubbles) provided by the purge linemay apply a force to the stuck cells greater than the one or more stiction forces. The force applied by the purge line fluid may be determined by one or more of a fluid flow rate, fluid density, fluid temperature, and fluid pressure. For example, the purge linemay introduce a fluid at a flow rate between about 1 mL/min and about 50 mL/min, about 1 mL/min and about 45 mL/min, or about 1 m/min and about 40 mL/min. For example, in some variations, the flow rate may be about 1 mL/min, about 5 mL/min, about 10 mL/min, about 15 m/min, about 20 mL/min, about 25 mL/min, about 30 mL/min, about 35 mL/min, about 40 mL/min, about 45 mL/min, or about 50 mL/min. The fluid may be introduced at a pressure between about 1 psi and about 15 psi, about 1 psi and about 12 psi, or about 1 psi and about 10 psi. For example, in some variations, the pressure may be about 1 psi, 2 psi, 3 psi, 4 psi, 5 psi, 6 psi, 7 psi, 8 psi, 9 psi, or 10 psi. In some variations, a volume of the fluid introduced by the purge linemay be between about 1 mL and about 150 mL, about 1 mL and about 120 mL, or about 6 mL and about 120 mL. For example, in some variations, the volume may be about 1 mL, about 6 mL, about 10 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, about 100 mL, about 110 mL, or about 120 mL.

The flow cellmay be configured to facilitate one or more measurements. For example, the flow cellmay be transparent such that one or more sensors (e.g., optical sensors, such as a camera) can measure one or more parameters of the cell solution contained within the flow cell. Accordingly, the flow cell, including fluid channeland/or film, may be manufactured from a material configured to facilitate the one or more measurements and/or withstand the environmental conditions (e.g., flow rates, pressures, temperatures) associated with the cell suspension. For example, the components of the flow cell, including the film, may be manufactured from a transparent material. In some variations, the material may comprise a plastic (e.g., polyethylene terephthalate glycol, polymethyl methacrylate) or a glass.

shows a rendering of an exemplary variation of a flow cellwithin a cell sorting module of a cartridge engaged with (e.g., coupled to) a magnetic arrayof a cell sorting instrument within a workcell. The flow cellmay comprise a fluid channel, an inlet port, a first outlet port, a second outlet port, and a gas port. The inlet portmay be positioned at a first end of the fluid channel. The gas portmay also be positioned at the first end of the fluid channel. The gas portmay be positioned such that any fluid (e.g., gas) introduced via the gas portmay flow past the inlet port. The positioning of the gas port relative to the inlet port may increase the efficacy of the fluid introduced via the gas port in removing any cellular material in any location within the fluid channel. The outlet ports,may be positioned at a second end of the fluid channel, where the second end is opposite the first end. Accordingly, fluid (e.g., cell suspension) may flow into the inlet port, along the entire fluid channel, and out of the outlet ports,. The fluid channelmay comprise a width. The widthmay correspond to a width of the magnetic arrayto facilitate even application of a magnetic field generated by the magnetic array, as will be described further herein.

shows an exemplary variation of the flow cellthat may not be engaged with a magnetic array. As shown, the flow cellmay comprise a filmconfigured to cover a fluid channel. The filmmay be configured to provide a fluid barrier between the fluid channeland an external environment, such that a cell suspension may flow through the fluid channelwithout leaking. Therefore, the filmmay advantageously have a thickness sufficient to withstand pressures, temperatures, and/or flow rates associated with the cell suspension without cracking, fracturing, shattering, or otherwise breaking, while still being configured to minimize a distance between the cell suspension flowing through the fluid channeland a magnetic array engaged with (e.g., coupled to) the flow cell. Accordingly, the filmmay comprise a thickness of between about 50 microns to about 500 microns, about 100 microns to about 500 microns, about 200 microns to about 400 microns, or about 250 microns to about 350 microns, including about 100 microns, about 200 microns, about 300 microns, or about 400 microns. The fluid channel, which may be covered by the film, may comprise a volume configured to contain the batch of cell suspension. For example, in some variations, the fluid channelmay be configured to hold a volume of fluid between about 0.5 mL to about 25 mL, about 1 mL to about 20 mL, or about 1 mL to about 15 mL, including about 1 mL, about 5 mL, about 8 mL, about 10 mL, about 12 mL, or about 15 mL. The batch of cell suspension may comprise a volume that may be the same as the fluid channel volume, or may be less. For example, in some variations, the batch of cell suspension may comprise a volume of about 0.5 mL to about 25 mL, about 1 mL to about 15 mL, about 6 mL to about 12 mL, or about 8 mL to about 10 mL, including about 5 mL, about 8 mL, about 9 mL, about 10 mL, or about 12 mL. Accordingly, the volume of cell suspension may be configured to avoid a buildup of pressure applied to the sidewall(s) of the flow cell, which may otherwise occur if the volume of cell suspension is greater than the volume of the flow cell.

show the flow cellcoupled to a plurality of fluid conduits (e.g., tubes), which may fluidically connect the flow cellto a fluidic manifold (not shown). The flow cellmay comprise a flow cell bodyconfigured to house the fluid channeland at least a portion of the fluid conduitsconnected to the flow cell, which may prevent damage to a connection point between a fluid conduit and the flow cell. As shown, the fluid conduitsof the flow cellmay comprise an inlet fluid conduitcoupled to the inlet port, a first outlet fluid conduitcoupled to the outlet port, a second outlet fluid conduitcoupled to the outlet port, and a purge linecoupled to the gas port. The inlet fluid conduitmay be configured to transfer a fluid, such as an unsorted cell suspension, into the fluid channelvia the inlet port. The outlet fluid conduits,may be configured to transfer a fluid, such as sorted components of the cell suspension, out of the fluid channelvia the outlet ports,. The purge linemay be configured to transfer a fluid (e.g., gas) into the fluid channelvia the gas port. For example, the purge linemay be used to introduce air into the fluid channelafter the non-targeted material of the cell suspension may be removed from the fluid channel, such that only targeted cells, including stuck cells, remain within the fluid channel. Accordingly, the air introduced via the purge linemay be configured to loosen the stuck cells, such that substantially all targeted cells may be removed from the fluid channel. For example, the purge linemay introduce air at a flow rate of between about 1 mL/min and about 40 mL/min and/or a pressure of between about 1 psi and about 10 psi. The volume of air introduced by the purge linemay be between about 6 mL and about 120 mL.

The fluid conduits,,,may each have dimensions (e.g., diameter, length, etc.) configured to facilitate fluid transfer to and/or from the flow cellin accordance with a pre-determined flow rate in accordance with a predetermined workflow. For example, a diameter of each fluid conduit,,,may be determined to minimize pressure drop of a fluid flowing therethrough. The diameter of each fluid conduit,,,may be the same or different as each other. In some variations, the diameter of each fluid conduit,,,may be between about 1 mm and about 4 mm. For example, in some variations, the diameter of one or more of the fluid conduits,,,may be about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, about 3 mm, about 3.5 mm, or about 4 mm. The flow rate either in or out of the flow cellmay be between about 1 mE/min to about 40 mL/min, about 3 mE/min to about 35 mL/min, or about 4 mE/min to about 30 mL/min, including about 4 m/min, about 10 mL/min, about 20 mL/min, or about 30 mL/min. In some variations, the cell suspension may be statically maintained within the flow cell. Statically maintaining the cell suspension within the flow cellmay advantageously facilitate increased efficacy of the cell sorting process. For example, a static cell suspension may reduce a drag force such that a magnetophoretic force applied by the magnetic array may be the greatest force applied to the cell suspension. Additionally or alternatively, statically maintaining the cell suspension may increase the period of time in which the targeted cells may be proximate to the magnetic array, such that a greater percentage of targeted cells may be attracted to the magnetic array. For example, the cell suspension may be maintained within the flow cellfor a period of between about 1 minute to about 10 minutes, about 2 minutes to about 8 minutes, or about 3 minutes to about 6 minutes, including about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10 minutes. The time period in which any portion of the cell suspension may be maintained within the flow cellmay correspond to the volume of the batch of cell suspension. That is, the time period may be proportional to the batch volume, such that increasing the batch volume may correspond to an increase in the time period and a decrease in the batch volume may correspond to a decrease in the time period.

illustrates an exemplary variation of a flow cellof a cell sorting module engaged with a magnetic arrayof a cell sorting instrument within a workcell. The flow cellmay comprise an inlet portand an outlet port. The flow cellmay comprise a lengththat may represent the distance between the inlet portand the outlet port. The lengthmay, in part, determine the quantity, strength, and/or spacing of the magnetsof the magnetic array, as will be described in further detail below. The magnetsmay comprise a magnet width (w). The flow cellmay comprise a heightthat may represent the height of the fluid channel. The heightmay correspond to the magnet width (w). That is, the heightmay be determined by the magnetic flux density of the magnetic field generated by the magnets, such that the magnetic flux density is sufficient at any location within the fluid channel to attract magnetic particles used in the cell sorting methods described herein. In some variations, the height of the flow cellmay be between about 1/16w to about ¼w, about 1/12w to about ⅛w, or about 1/11w to about 1/9w, including about 1/12w, about 1/10w, or about ⅛w.

Furthermore, the heightmay advantageously accommodate magnetic particles that may be micrometer-sized and/or nanometer-sized. That is, the height of the fluid channel may be such that the magnetic particles may flow through the fluid channel without sticking to the sidewalls defined by the fluid channel and/or the film, which may prevent the magnetic particles from clogging or otherwise restricting fluid flow through the fluid channel. The height may also facilitate rinsing, washing, and/or purging the flow cell by providing adequate volume around any cells that may be stuck within the flow cell due to one or more stiction forces. In some variations, the height may also facilitate high cell processing throughput by accommodating relatively high flow rates, various magnetic particle sizes, and/or fluid volumes, which may advantageously provide flexibility such that advanced knowledge of the desired throughput of targeted cells may not be required. Additionally, the height may also be as small as possible to maximize the effect of the magnetic field in attracting targeted cells. That is, it may be advantageous to flow the fluid within the flow cell as close as possible to the magnetic array to attract the targeted cells. For example, the flow cellmay comprise a height of between about 0.25 mm to about 5 mm, about 1 mm to about 3 mm, or about 1 mm to about 2 mm, including about 0.25 mm, about 1 mm, about 1.5 mm, about 2 mm, about 2.5 mm, about 3 mm, about 3.5 mm, about 4 mm, about 4.5 mm, or about 5 mm.

The cell sorting modules within a cell processing cartridge as described herein correspond to a cell sorting instrument configured to perform cell sorting within a cell processing workcell. For example, the workcell may comprise a bay having a cell sorting instrument with one or more components that may interact or releasably couple to the cell sorting module of the cartridge to perform a cell sorting process. The one or more components of the cell sorting instrument may include a magnetic array having one or more magnets, which may be configured to generate a magnetic field configured to attract magnetic particles coupled to targeted cells (e.g., magnetically tagged cells). The magnetic array may be movable and/or releasably couplable to the cell processing module within the cartridge, such that the magnetic field may be selectively applied to targeted cells flowing through a flow cell of a cell sorting module. In some variations, the magnetic field may be substantially uniform across the flow cell, such that the targeted cells may be attracted to the magnetic array at any location within the flow cell.

Referring to, a block diagram of an exemplary variation of a cell sorting instrumentis shown. The cell sorting instrumentmay comprise components configured to perform cell sorting. As shown, the cell sorting instrumentmay comprise a magnetic array, an actuator, a sensor, and a prism. The magnetic arraymay comprise one or more magnets configured to generate a magnetic field. The one or more magnets may be permanent magnets, temporary magnets, and/or electromagnets. The magnetic field generated by the magnetic arraymay be configured to attract magnet particles, such as the magnetic particles coupled to targeted cells in a cell suspension. The magnets of the magnetic arraymay be arranged to be coplanar with each other. Systems that utilize a magnetic array with a three-dimensional arrangement of magnets, such as a U-shaped arrangement, may require manually coupling the magnetic array to a fluid container that may contain a cell suspension. Accordingly, the coplanar arrangement utilized by the magnetic arraydescribed herein may facilitate automation, as coupling the magnetic arrayto the flow cellmay be achieved by contact between the planar magnetic array and the planar flow cell.

The magnets of the magnetic arraymay be arranged such that the magnetic field generated by the magnets extend through substantially all of the fluid channeldescribed in reference to. Accordingly, a targeted cell in any portion of the fluid channelmay experience a magnetic field with a strength sufficient to attract the targeted cell towards the magnetic array. The magnetic arraymay be coupled to the actuator. The actuatormay be configured to move the magnetic array. For example, the actuatormay translate the magnetic arrayin a direction perpendicular to the flow cell. The translation of the magnetic arrayby the actuatorhelps to engage (e.g., couple) and/or disengage (e.g., decouple) the magnetic arrayfrom the flow cells described herein. For example, the actuatormay translate the magnetic arrayin a first direction (e.g., extend the actuator) to engage the magnetic arrayto the flow cell, and may translate the magnetic arrayin a second opposite direction (e.g., retract the actuator) to disengage the magnetic arrayfrom the flow cell. When the magnetic arrayis engaged with the flow cell, the magnetic field generated by the magnetic arraymay interact with the cell suspension flowing through the fluid channel. When the magnetic arrayis disengaged from the flow cell, the magnetic field generated by the magnetic arraydoes not interact with the cell suspension flowing through the fluid channel. The magnetic array may comprise one or more magnets to generate a magnetic field of sufficient strength to perform the cell sorting processes described herein. For example, in some variations, the magnetic arraymay comprise between 1 and 30 magnets, 1 and 20 magnets, 5 and 20 magnets, or 7 and 17 magnets, including 1 magnet, 5 magnets, 7 magnets, 10 magnets, 15 magnets, or 17 magnets. The one or more magnets of the magnetic arraymay comprise a magnetic material, such as a metal (e.g., iron, cobalt, nickel, samarium, neodymium, or an alloy thereof). For example, the magnets may comprise neodymium of a grade (e.g., magnetic strength) between N35 and N55 or N35 and N52. In some variations, the neodymium may comprise a grade of N35, N38, N40, N45, N48, N50, N52, or N55. In some variations, the one or more magnets may be permanent magnets, such that a magnetic field may be permanently generated. In further variations, the one or more magnets may be electromagnet(s), such that a current may be used to control the magnetic field. That is, the current may be proportional to the strength of the magnetic field. For example, a current between about 0 A to about 10 A may be applied to the electromagnet(s). Accordingly, one or more wires may be connected to the magnetic arraysuch that an electrical signal may be provided to the magnetic array. The current provided via the electrical signal may be controlled by a controller, such as the controllerdescribed in reference to. In still further variations, the magnetic arraymay comprise a mix of permanent magnets and electromagnets, such that the magnetic field may be increased by applying a current, if necessary to perform the cell sorting process.

Any number and type of sensor may be used with the cell sorting modules and cell sorting instruments described herein. For example, instrument sensormay be configured to measure one or more parameters associated with the cell suspension, such as cell count value, cell density value, cell size value, flow rate, pH, and/or a dissolved oxygen value. In some variations, the sensormay comprise an optical sensor, such as a camera. The instrument sensormay be positioned adjacent flow cell. In some variations, the instrument sensormay utilize one or more mirrored surfaces to define an optical path between the instrument sensorand the flow cell. For example, a prismmay be configured to provide an optical path through or around the cell sorting module. The optical path provided by the prismmay be necessary due to the position of the instrument sensorrelative to the flow cell. That is, the instrument sensormay not have a direct line-of-sight to the flow cellbecause other components of the cartridge may prevent directly coupling the instrument sensorto the flow cell. Accordingly, the prismmay comprise one or more mirrors configured to provide an optical path between the sensorand the flow cell. For example, the one or more mirrors may provide an optical path around one or more components that would otherwise prevent the instrument sensorfrom observing the flow cell. The one or more mirrors may reflect light such that the optical path travels around one or more corners. In some variations, the prismmay comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mirrors. The mirror(s) of the prismmay have any suitable shape e.g., a square, a rectangle, a circle, or a combination thereof. The mirror(s) of the prismmay comprise a material configured to provide a mirrored surface, such as a glass, a metal, or a combination thereof. The optical path will be described in further detail below, particularly in reference to.

illustrate exemplary variations of a flow cellengaged with a cell sorting instrument. The cell sorting instrumentmay comprise a magnetic array, a piston, guides,, an array housing, a cameracomprising a lens, a camera support, and mirrors,. The pistonmay be coupled to the magnetic array. The pistonmay be configured to move the magnetic arraybetween a retracted configuration and an extended configuration.shows the magnetic array in the retracted configuration, such that the magnetic arraymay be received within the array housing. When in the retracted configuration, the magnetic arrayis disengaged from the flow cellso that no cell sorting is performed.

shows the pistonand magnetic arrayin the extended configuration. In the extended configuration, the magnetic array is proximate to the flow celland the magnetic field may be applied to targeted cells therein. The magnetic arraymay move between the extended and retracted configurations via the pistonand guides,, which will be described in further detail below in reference to.

also shows cameracoupled to the array housingvia the camera support. The camera supportmay be coupled to the array housingadjacent a first end of the array housing. The flow cellmay be positioned adjacent a second end of the array housing. Accordingly, the cameramay not have a direct line-of-sight to the flow cell. Therefore, the mirrors,may define a prism configured to provide an indirect line-of-sight between the cameraand the flow cell. The line-of-sight may be referred to as an optical path.

illustrates an exemplary variation of an optical pathfrom the camerato the flow cell. Due to spatial constraints imposed by the cell sorting instrument, the cameramay be positioned offset from the flow cellsuch that the cameramay not have a direct line-of-sight to the flow cell. Accordingly, the cameramay utilize one or more prisms comprising one or more mirrors to reflect light from the flow cellto the camera, as described above. As shown, the camera, via the lens, may be pointed at the first mirror. The first mirrormay be angled relative to the camera. For example, the first mirrormay be angled relative to the camerabetween about 5 degrees to about 85 degrees, about 20 degrees to about 60 degrees, about 30 degrees to about 60 degrees, or about 40 degrees to about 50 degrees, including about 40 degrees, about 45 degrees, or about 50 degrees. In some variations, the first mirrormay be angled such that an optical path from the cameramay be redirected towards the second mirror. The second mirrormay be angled relative to the flow cell. For example, the second mirrormay be angled relative to the flow cellbetween about 5 degrees to about 85 degrees, about 20 degrees to about 60 degrees, about 30 degrees to about 60 degrees, or about 40 degrees to about 50 degrees, including about 40 degrees, about 45 degrees, or about 50 degrees. The first and second mirrors,may be parallel to each other. In some variations, the first and second mirrors,may be angled relative to each other. The angle of the first and/or second mirrors,relative to the cameraand/or flow cellmay be determined by the position of the camera, the second mirror, and/or the flow cell.

The cameramay be configured to generate a clear image of any location within the flow cell, such that the cameramay have a focal length corresponding to a total distance between the cameraand the flow cell. For example, the first mirrormay be separated from the lensby a distance, such as between about 12.5 mm to about 75 mm, including about 25 mm. The second mirrormay be separated from the first mirrorby a distance, such as between about 12.5 mm to about 150 mm, including about 75 mm. The second mirrormay be separated from the flow cellby a distance, such as between about 12.5 mm to about 50 mm, including about 12.5 mm. Accordingly, the lensmay comprise a focal length corresponding to a total distance between the lensand the flow cell. The total distance between the lensand the flow cellmay include the distance between the first mirrorand the lens, the first mirrorto the second mirror, and the first mirrorto the flow cell. In some variations, the total distance may be between about 25 mm to about 250 mm, including about 25 mm, about 50 mm, about 75 mm, about 100 mm, about 125 mm, or about 150 mm.